一株辛德毕斯病毒的基因组序列测定及分析

Genome Sequencing and Analysis of one Sindbis Virus Strain

目的:对引进的一株辛德毕斯病毒的基因组序列进行测定,阐明其与已报道毒株序列的关系。方法:对辛德毕斯病毒基因组编码区进行分段RT-PCR扩增,对非编码区采用RACE法进行扩增,将扩增产物直接进行测序,应用DNAStar软件将测序结果拼接得到基因组序列,采用MEGA3.1软件对9株辛德毕斯病毒基因组序列进行系统进化发生树的构建。结果与结论:此株辛德毕斯病毒基因组共11663nt,编码3745个氨基酸残基,其中5'端的2/3基因组编码4种非结构蛋白NSp1、NSp2、NSp3和NSp4,3'端的1/3基因组编码5种结构蛋白E1、E2、E3、6K和C;结构基因和非结构基因之间有48nt的连接区为非翻译区;病毒基因组5'末端和3'末端分别有59、318nt的非编码区;序列同源性分析结果表明,此株病毒与S.A.AR86株的同源性最高,两者核苷酸序列的同源性为99.7%,氨基酸序列的同源性为99.6%,而与本室保存的另一辛德毕斯病毒MEI株的遗传进化关系稍远,系统进化发生树处于不同分支上。

Objective： To analyze the genome sequence of a Sindbis virus strain WJ8601 and reveal its genome differences with other reported Sindbis virus strains. Methods： The coding sequences were amplified by 13 different RT-PCR reactions, and the noneoding region was amplified by RACE method and the sequenced eDNA fragments were analyzed by DNAStar software and assembled to a complete genome. Phylogenetie and molecular evolutionary analyses were conducted using MEGA version 3.1. We performed a phylogenetie analysis and generated a neighbor-joining tree based on the com- plete genomic sequences of WJ8601 and 8 Sindbis viruses available in GenBank. Results ＆ Conclusion： This WJ8601 strain has a genome of 11 663 nt, as the common genome of Sindbis virus, which encodes 3 745 amino acids. The 5 terminal two-thirds of RNA encodes four nonstructural proteins, NSp1, NSp2, NSp3 and NSp4. The 3 terminal one-third of RNA encodes five structural proteins, El, E2, E3, 6K and C. A 48 nt untranslated region connected the nonstructural and the structural protein coding sequences. The noneoding regions was constituted of 59 nt at 5＂ terminal and 318 nt at 3＂ terminal. Sequence comparison showed that the WJ8601 strain has a nueleotide sequence identity of 99.7% and an amino acid sequence identity of 99.4% with S.A.AR86 strain. Phylogenetic analysis demonstrated WJ8601 strain has closest relation with S.A.AR86 strain and has closer relation with MEI strain. Phylogenetic tree showed that WJ8601 strain and MEI strain clustered into different branch.